Changing the lactose permease of Escherichia coli into a galactose-specific symporter
Changing the lactose permease of Escherichia coli into a galactose-specific symporter
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DOI：
10.1073/pnas.102178299
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发表时间：
2002-05-14
影响因子：
11.1
通讯作者：
Kaback, HR
中科院分区：
文献类型：
--
作者：
Guan, L;Sahin-Tóth, M;Kaback, HR
N-乙基马来酰亚胺(NEM)修饰的乳糖渗透酶突变体含有单一半胱氨酸，取代了Ala-122(螺旋IV)，取消了乳糖的主动转运。此外，乳糖、蜜二糖和β，D-半乳糖基1-硫代，D-半乳糖苷可保护NEM抑制[C-14]NEM对Cys-122的乳糖转运和/或烷基化。然而，值得注意的是，D-半乳糖的运输相对不受NEM的影响，而且单糖对NEM失活乳糖运输没有任何保护作用。NEM修饰后，乳糖、蜜二糖或β，D-半乳糖基1-硫代-D-半乳糖苷对[C-14]半乳糖转运的竞争性抑制作用显著降低，而未标记的半乳糖对[C-14]半乳糖转运的抑制作用不受影响。结果表明，Cys-122的烷基化选择性地抑制了双糖的结合和转运，而单糖半乳糖的转运基本不受影响。此外，虽然保守突变Ala-122-->；Ser仅引起乳糖转运的轻微抑制，但突变Ala-122-->；Phe和Ala-122-->；Tyr会导致明显的抑制。与之形成鲜明对比的是，这些替代品对半乳糖的转运都没有显著影响。结果表明，Ala-122是配体结合部位的一个组成部分，并提供了一个强烈的迹象，即122位的侧链紧靠在D-半乳糖苷的非半乳糖基团上。这与Cys-148形成了鲜明的对比，Cys-148是螺旋V中的一个邻近残基，它与D-半乳糖苷的半乳糖部分的疏水面相互作用。
N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [C-14]NEM. Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [C-14]galactose transport by lactose, melibiose, or beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. in addition, although the conservative mutation Ala-122 --> Ser causes only mild inhibition of lactose transport, the mutations Ala-122 --> Phe and Ala-122 --> Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the nongalactosyl moiety Of D-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides.